One known test procedure or process for detection of a specific bioagent is the enzyme linked immunoassay (hereafter referred to as “ELISA”). The ELISA process is an identification process that uses molecular interactions to uniquely identify target substances and is applicable to a variety of fields, such as biotechnology, environmental protection and public health. A basic definition of ELISA is a quantitative in vitro test for an antibody or antigen (e.g. a bioagent) in which the test material is adsorbed on a surface and exposed to a complex of an enzyme linked to an antibody specific for the substance being tested for with a positive result indicated by a treatment yielding a color in proportion to the amount of antigen or antibody in the test material. The basic ELISA procedure is described more specifically, for one, in a book entitled, Methods in Molecular Biology, Vol. 42, John R. Crowther, Humana Press, 1995.
The “antibody specific for the substance being tested for” in the foregoing definition constitutes a recognition molecule, a molecule that is capable of binding to either reactant or product molecules in a structure-restricted manner. That is, the recognition molecule binds to a specific three-dimensional structure of a molecule or to a two-dimensional surface that is electrically charged and/or hydrophobic in a specific surface pattern. It may also be recognized that ELISA-like approaches using other recognition molecules can also be used, such as aptamers, DNA, RNA and molecular imprint polymers.
More recently, the foregoing definition of ELISA has been expanded beyond the colormetric approach, in which color and color intensity is used as the reporter or indicia of the antigen or antibody, to include a voltametric or amperiometric approach to detection and assay, in which the rate of change of voltage or current conductivity is proportional to the amount of antigen or antibody contained in the test material. Patent Cooperation Treaty application PCT/US98/16714, filed Aug. 12, 1998 (International Publication No. WO 99/07870) entitled, “Electrochemical Reporter System for Detecting Analytical Immunoassay and Molecular Biology Procedures” (hereafter the “16714 PCT application”), claiming priority of U.S. patent applications Ser. Nos. 09/105,538 and 09/105,539), to which the reader may refer, describes both a colormetric and an electrochemical reporter system for detecting and quantifying enzymes and other bioagents in analytical and clinical applications. The electrochemical reporter system of the 16714 PCT application employs a sensor for detecting voltametric and/or amperiometric signals that are produced in proportion to the concentration of organic (or inorganic) reporter molecules by redox (e.g. reduction-oxidation) recycling at the sensor.
In brief, in the ELISA test, the suspect bioagent is initially placed in a water-based buffer, such as a phosphate buffered saline solution, to form a sample solution. That sample solution is mixed with a quantity of particles, beads, the surface of which is coated with an antibody to the suspect bioagent, a recognition molecule (also sometimes referred to as a receptor molecule). The particular antibody used to coat the beads is known to bind to the bioagent of interest and is a primary antibody or “1° Ab.” That is, the antibody coating exhibits a chemical “stickiness” that is selective to specific bioagents.
Any bioagent that is present in the sample solution binds with a non-covalent bond to a respective antibody and thereby becomes attached to a respective one of the beads in the mixture-solution. If the sample solution does not contain a bioagent or if the bioagent that is present in the solution is not one that binds to the selected antibody, then nothing binds to the foregoing antibody. Further processing of the ELISA process then shows nothing.
Assuming the suspect bioagent is present in the sample, the bioagent bonds to the antibody that is coated on the beads. The solution then contains a quantity of bioagent molecules bound respectively to a quantity of coated beads. The mixture is optionally washed, as example, in a phosphate-buffered saline, and a second antibody, more specifically, an antibody and enzyme linked combination, is then added to the mixture. The second antibody is also one that is known to bind to the suspect bioagent, another recognition molecule. The second antibody may either be one that is monoclonal, e.g. one that binds to only one specific molecule, or polyclonal, e.g. a mixture of different antibodies each of which shares the characteristic of bonding to the target bioagent. The enzyme is covalently bound to the second antibody and forms a complex that is referred to as a secondary antibody-enzyme conjugate or “2° Ab-enz.” As is known, an enzyme is a “molecular scissors,” a protein that catalyzes a biological reaction, a reaction that does not occur appreciably in the absence of the enzyme. The enzyme is selected to allow the subsequent production of an electrochemically active reporter.
The 2° Ab-enz binds to the exposed surface of the immobilized bioagent to form an “antibody sandwich” with the bioagent forming the middle layer of that sandwich. The antibody sandwich coated beads are washed again to wash away any excess 2° Ab-enz in the solution that remains unbound.
In the process of the 16714 PCT application, the beads and the attached antibody sandwich (e.g. the 1° Ab/bioagent/2° Ab-enz complex) in the solution are placed over the exposed surface of the electrical redox recycling sensor. The substrate of the foregoing enzyme, referred to as PAP-GP (e.g. p-nitrophenyl beta-D-galactopyranoside), is added to the solution. The substrate is cleaved by the enzyme to produce an electrochemically active reporter. The substrate of the enzyme is any substance that reacts with an enzyme to modify the substrate. The effect of the enzyme is to separate, cut, the PAP, a para-amino phenol, the electrochemically active reporter, from the GP, an electrochemically inactive substance.
The foregoing chemical reaction is concentrated at the surface of the sensor. The rate of production of the foregoing reporter (e.g. the PAP) is proportional to the initial concentration of bioagent. The reporter reacts at the surface of the sensor, producing an electrical current through the sensor that varies with time and is proportional to the concentration of the bioagent, referred to as redox recycling. The occurrence of the electric current constitutes a positive indication of the presence of the suspect bioagent in the sample. Analysis of the electric currents produced over an interval of time and comparison of the values of that electric current with existing laboratory standards of known bioagents allows quantification of the concentration of bioagent present in the initial sample.
Both of the foregoing ELISA processes are carried out manually in the laboratory by skilled personnel. In the automated apparatus disclosed in our U.S. application, Ser. No. 09/837,946, filed Apr. 19, 2001 entitled, “Automated Computer Controlled Reporter Device for Conducting Immunoassay and Molecular Biology Procedures” (the “'946 application”), hereafter sometimes referred to as the automated ELISA system, a user friendly stand-alone portable system is disclosed that automatically performs the ELISA process. The automated ELISA system contains a number of solutions in respective reservoirs and pumps that are controlled by a programmed computer. The electronic controller, such as a programmed microcontroller, controls a series of electric pumps to automatically sequence the pumping of the individual solutions required by the ELISA procedure into and out of a cell (or cells) as required by the ELISA program. The controller commands the steps necessary to produce the reporter, controls the positioning of the carrier of the reporter adjacent the reporter sensor, analyzes the data obtained from the reporter sensor and displays the concentration of the bioagent determined from the analysis of the foregoing data. Once started, the apparatus, governed by the program, conducts the test automatically without the necessity for human intervention. In that apparatus, the controller is able to manipulate the position of the 1° antibodies in the washing step and the position the antibody sandwich formed in the later steps of the process to the reporter sensor with magnetic fields by attaching the 1° antibodies to magnetic beads.
In a first step of the automated assay procedure, the sample solution, containing the sample that is to be tested for the presence of a specific bioagent, is placed in a container (or equivalent vessel) that holds the 1° antibody coated magnetic beads. If the sample is of the specific bioagent, then the respective parts of the sample links sticks to the antibody coating of a respective bead. As example, the sample solution that is to be tested for the presence of a specific bioagent and the coated magnetic beads are pumped from respective reservoirs into a container by electrical pumps and mixed to ensure that the respective parts, that is, molecules, of the sample contacts the coating of a respective bead. Some of the sample may be unattached to a bead and that excess needs to be removed from the solution by washing. To wash the mixture, the magnetic beads (and attached molecules) are pulled to one side of the container by a magnetic field controlled by the controller, vacating a portion of the solution. The controller causes the pumps to remove the dirty solution through an aspirating pipe immersed in that vacated portion of the solution and to replace the dirty solution with clean solution, effectively washing the bioagent/antibody complex in the vessel. In the later step of the automated process, the controller draws the formed 1° Ab/bioagent/2° Ab-enz complex to the reporter sensor prior to pumping the substrate of the enzyme into the solution, whereby the reporter developed by cleavage of the substrate is properly positioned for monitoring by the sensor.
Because the process is automated, the testing is typically accomplished more quickly than the prior manual processes. Even so, the automated process requires a finite interval to complete. Awaiting the outcome of the test procedure is always somewhat stressful, a period of anxiousness in contemplation of the uncertainty of the outcome, and to the impatient person that testing interval, though short, may seem to last forever. Reducing the time to complete the automated test is seen, for one, as a way to reduce that stress on the user or, if completed very quickly, eliminate the lag time during which that stress develops. As an advantage, the invention reduces the testing period of the automated process.
Accordingly, an object of the present invention is to reduce the time required to perform an ELISA procedure.
Another object of the invention is to provide an automated reporter system for performing an ELISA procedure in a shorter period of time than was previously possible and at less expense.